Titanium Dioxide Nanoparticles Modulate Systemic Immune Response and Increase Levels of Reduced Glutathione in Mice after Seven-Week Inhalation

Titanium dioxide nanoparticles (TiO2 NPs) are used in a wide range of applications. Although inhalation of NPs is one of the most important toxicologically relevant routes, experimental studies on potential harmful effects of TiO2 NPs using a whole-body inhalation chamber model are rare. In this study, the profile of lymphocyte markers, functional immunoassays, and antioxidant defense markers were analyzed to evaluate the potential adverse effects of seven-week inhalation exposure to two different concentrations of TiO2 NPs (0.00167 and 0.1308 mg TiO2/m3) in mice. A dose-dependent effect of TiO2 NPs on innate immunity was evident in the form of stimulated phagocytic activity of monocytes in low-dose mice and suppressed secretory function of monocytes (IL-18) in high-dose animals. The effect of TiO2 NPs on adaptive immunity, manifested in the spleen by a decrease in the percentage of T-cells, a reduction in T-helper cells, and a dose-dependent decrease in lymphocyte cytokine production, may indicate immunosuppression in exposed mice. The dose-dependent increase in GSH concentration and GSH/GSSG ratio in whole blood demonstrated stimulated antioxidant defense against oxidative stress induced by TiO2 NP exposure.


Introduction
Titanium dioxide nanoparticles (TiO 2 NPs) are one of the most widely produced nanomaterials in the world. They are used in a wide range of applications, including coatings, paints, plastics, paper, inks, textiles, cosmetics, pharmaceuticals, food, agricultural production, environmental remediation, wastewater treatment, antibacterial agents, electronics, catalysts, solar cells, biomedical applications, etc. [1,2]. The mass production stress may cause inflammation, which might be stimulated by activation of ROS signaling pathways [25]. The results of a recent study support the theory that TiO 2 NPs induce the formation of ROS and inhibit cellular enzymatic mechanisms, including effects on GSH levels [26].
Numerous in vitro and in vivo studies have demonstrated the potential of TiO 2 NPs for immunomodulatory effects [27][28][29][30][31][32]. However, only a few have studied the effects of inhalation exposure on the systemic immune response [8,33,34]. Inhaled TiO 2 NPs have been found to affect the number of immune cells in the blood, such as monocytes, granulocytes, lymphocytes, and platelets [7,33]. They are readily taken up by cells of the immune system, can accumulate in peripheral lymphoid organs such as the spleen and lymph nodes, and influence various manifestations of immune cell activity, including cytokine production [18,34,35] and proliferation [8,35].
In this study, we investigated the potential effects of TiO 2 NPs on immune response and antioxidant defense. Here, we report the effects of subchronic inhalation exposure to two different concentrations of these NPs (0.00167 and 0.1308 mg TiO 2 /m 3 ) carried out in whole-body inhalation chambers continuously for seven weeks. Although several inhalation studies on the systemic immune effects of TiO 2 -NPs have been conducted previously, they were performed by intratracheal [8,33,35] or intranasal [34] instillation. Compared with instillation, inhalation better mimics the physiological pathway of inhalation exposure to nanomaterials.
Because reliable data from studies with doses relevant to human exposure are still lacking, the concentrations of TiO 2 NPs used in the present study were lower than in previous studies, i.e., closer to a realistic exposure scenario in occupational settings [9,10].
The aim of our study was to investigate the effects of TiO 2 NPs on the systemic immune response and antioxidant defense in the blood of mice (GSH, GSSG, and GSH/GSSG ratio) after seven weeks of continuous inhalation.

Animals
Adult female mice (ICR line, six weeks old, average body weight 24 g) were obtained from Masaryk University (Brno, Czech Republic). Prior to the experiment, the animals were acclimated to laboratory conditions for one week. A commercial diet and water were provided ad libitum. All animal experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Institute of Analytical Chemistry of the Czech Academy of Sciences (Ministry of Agriculture of the Czech Republic, No. 10031/2013-MZE-17214) and approved by the Animal Ethics Committee of the Institute of Animal Physiology and Genetics of the Czech Academy of Sciences (No. 081/2010).

Preparation of NPs
TiO 2 NPs were continuously generated in situ via the aerosol route in a hot-wall tubular flow reactor by thermal decomposition of titanium tetraisopropoxide (TTIP) in a vertically oriented furnace (Carbolite TZF 15/50/610) at a temperature of 751 • C [46]. The vapors of TTIP were generated from the liquid form of TTIP in a saturator at 24 • C and the released vapors were transported into the flow reactor with a nitrogen stream (purity 99.9995%; flow rate 0.85 L/min). Before entering the reactor, it was diluted with another nitrogen stream (purity 99.9995%; flow rate 0.90 L/min). In parallel, a stream of oxygen (99.9996%; flow rate 0.40 L/min) was introduced into the reactor to oxidize the organic part of the TTIP. At the outlet of the reactor, the TiO 2 NPs transported in a mixture Nanomaterials 2023, 13, 767 4 of 16 of nitrogen and oxygen (flow rate 2.15 L/min) were mixed with air (flow rate 5.00 L/min). The generated TiO 2 NPs were consecutively diluted with U-HEPA-filtered air (flow rate 20.0 L/min) and used for whole-body inhalation experiments in two inhalation cages with different concentrations of TiO 2 NPs. All flow rates were regulated with Aalborg GFCS electronic mass flow controllers.
The size and shape of the generated TiO 2 NPs were characterized by transmission electron microscopy (TEM, Magellan 400 L XHR microscope, FEI Company, Hillsboro, OR, USA). TiO 2 NPs were collected by electrostatic precipitation by using a nanometer aerosol sampler (Model 3089, TSI, Shoreview, MN, USA) on TEM grids (copper S160-4, 3 mm diameter, 400 mesh grid, Agar Scientific, Electron Technology, Stansted, Essex, UK). Samples were analyzed by scanning transmission electron microscopy in STEM mode. The micrograph (Figure 1) showed that the TiO 2 NPs in the air inside the inhalation cage, measured by a Scanning Mobility Particle Sizer (model 3972L, TSI, USA), consisted mainly of agglomerates of primary particles with diameters ranging from 2 to 6 nm and small number of larger particles with size up to a diameter of 15 nm was also found. released vapors were transported into the flow reactor with a nitrogen stream (purity 99.9995%; flow rate 0.85 L/min). Before entering the reactor, it was diluted with another nitrogen stream (purity 99.9995%; flow rate 0.90 L/min). In parallel, a stream of oxygen (99.9996%; flow rate 0.40 L/min) was introduced into the reactor to oxidize the organic part of the TTIP. At the outlet of the reactor, the TiO2 NPs transported in a mixture of nitrogen and oxygen (flow rate 2.15 L/min) were mixed with air (flow rate 5.00 L/min). The generated TiO2 NPs were consecutively diluted with U-HEPA-filtered air (flow rate 20.0 L/min) and used for whole-body inhalation experiments in two inhalation cages with different concentrations of TiO2 NPs. All flow rates were regulated with Aalborg GFCS electronic mass flow controllers.
The size and shape of the generated TiO2 NPs were characterized by transmission electron microscopy (TEM, Magellan 400 L XHR microscope, FEI Company, Hillsboro, OR, USA). TiO2 NPs were collected by electrostatic precipitation by using a nanometer aerosol sampler (Model 3089, TSI, Shoreview, MN, USA) on TEM grids (copper S160-4, 3 mm diameter, 400 mesh grid, Agar Scientific, Electron Technology, Stansted, Essex, UK). Samples were analyzed by scanning transmission electron microscopy in STEM mode. The micrograph (Figure 1) showed that the TiO2 NPs in the air inside the inhalation cage, measured by a Scanning Mobility Particle Sizer (model 3972L, TSI, USA), consisted mainly of agglomerates of primary particles with diameters ranging from 2 to 6 nm and small number of larger particles with size up to a diameter of 15 nm was also found.
The surface areas of the generated TiO2 NPs were 1.94 × 10 8 and 1.16 × 10 10 nm 2 /cm 3 . The surface area was calculated from the NP size distribution data by using SMPS software [47].

Exposure to TiO2 NPs
Animals were exposed to TiO2 NPs in a special inhalation chamber [48], which was made of glass and stainless steel and contained four stainless steel inhalation cages. A control group of animals was housed in the inhalation chamber in a cage without exposure to NPs. The air conditioning system maintained constant parameters of the air flowing through the inhalation cages (i.e., temperature, relative humidity, and pressure). Air parameters were measured and recorded online at one-minute intervals. Lighting was set to 12 h of light and 12 h of darkness. The behavior and health status of the mice were continuously monitored with a camera system. The distributions of generated NPs were meas- The surface areas of the generated TiO 2 NPs were 1.94 × 10 8 and 1.16 × 10 10 nm 2 /cm 3 . The surface area was calculated from the NP size distribution data by using SMPS software [47].

Exposure to TiO 2 NPs
Animals were exposed to TiO 2 NPs in a special inhalation chamber [48], which was made of glass and stainless steel and contained four stainless steel inhalation cages. A control group of animals was housed in the inhalation chamber in a cage without exposure to NPs. The air conditioning system maintained constant parameters of the air flowing through the inhalation cages (i.e., temperature, relative humidity, and pressure). Air parameters were measured and recorded online at one-minute intervals. Lighting was set to 12 h of light and 12 h of darkness. The behavior and health status of the mice were continuously monitored with a camera system. The distributions of generated NPs were measured directly in the inhalation cages with a scanning mobility particle sizer. The size distribution is shown in Figure 2 for low and high concentrations of TiO 2 NPs, respectively. The number concentrations of TiO 2 NPs were 5.06 × 10 4 (mode 32.2 nm, geometric mean diameter 29.6 nm, geometric standard deviation 1.64) and 1.51 × 10 6 particles/cm 3 (mode 37.2 nm, geometric mean diameter 30.3 nm, geometric standard deviation 1.85). The average mass concentrations of TiO 2 NPs were 1.67 and 130.8 µg TiO 2 /m 3 for low and high number concentrations, respectively. The special feeding device (a tube closed at the top from which the feed falls down into the feeder) was designed to minimize oral ingestion of NPs NPs on their surface. Mice were exposed to TiO2 NPs continuously for seven weeks, 24 h/day, seven days/week. Control animals were exposed to the same air as the experimental groups, only without the NP supplement. The estimated total deposited dose during the sevenweek inhalation period was 0.012 and 0.958 µg TiO2 per gram of body weight of mice [49]. At the end of the inhalation experiment, the mice were directly decapitated and immediately dissected. Blood and organs were isolated and subjected to immune system and antioxidant defense examination.

Figure 2.
The number size distribution of TiO2 NPs for low NP concentration (5.06 × 10 4 particles/cm 3 ) and high NP concentration (1.51 × 10 6 particles/cm 3 ). x-axis, particle diameter (a logarithmic scale); y-axis, number concentration of particles (the number concentration is normalized by the size range of particles)

Phagocytic Activity of Granulocytes and Monocytes and Respiratory Burst of Phagocytes
The assay was performed as described elsewhere [50]. Heparinized whole blood from mice was mixed with hydroethidine and incubated at 37 °C for 15 min. Subsequently, samples were incubated with fluorescein-labeled Staphylococcus aureus (SA, Invitrogen, Waltham, MA, USA) for an additional 15 min at 37 °C. Then they were placed on Figure 2. The number size distribution of TiO 2 NPs for low NP concentration (5.06 × 10 4 particles/cm 3 ) and high NP concentration (1.51 × 10 6 particles/cm 3 ). x-axis, particle diameter (a logarithmic scale); y-axis, number concentration of particles (the number concentration is normalized by the size range of particles). Mice were exposed to TiO 2 NPs continuously for seven weeks, 24 h/day, seven days/week. Control animals were exposed to the same air as the experimental groups, only without the NP supplement. The estimated total deposited dose during the seven-week inhalation period was 0.012 and 0.958 µg TiO 2 per gram of body weight of mice [49]. At the end of the inhalation experiment, the mice were directly decapitated and immediately dissected. Blood and organs were isolated and subjected to immune system and antioxidant defense examination.

Phagocytic Activity of Granulocytes and Monocytes and Respiratory Burst of Phagocytes
The assay was performed as described elsewhere [50]. Heparinized whole blood from mice was mixed with hydroethidine and incubated at 37 • C for 15 min. Subsequently, samples were incubated with fluorescein-labeled Staphylococcus aureus (SA, Invitrogen, Waltham, MA, USA) for an additional 15 min at 37 • C. Then they were placed on ice and cold lysis solution was added. For the control tubes, the SA bacteria were added after the lysis solution. EPICS XL flow cytometer (Beckman Coulter, Brea, CA, USA) was used to analyze the samples by using forward and side scatter gates. The percentage of phagocytic monocytes, phagocytic granulocytes, and granulocytes with respiratory burst was measured in duplicate. The results were analyzed by flow cytometry as follows: % of phagocytic granulocytes = phagocytic granulocytes/all granulocytes.

In Vitro Production of Cytokines and Chemokines
Spleen cells were cultured with the mitogen concanavalin A

Antioxidant Status, Reduced Glutathione, and Oxidized Glutathione
Concentrations of reduced (GSH) and oxidized (GSSG) glutathione as markers of oxidative stress and antioxidant defense were determined by using the Bioxytech ® GSH/GSSG-412™ assay kit (Oxis International, Inc., Portland OR, USA) according to the method of Ellman [51], modified by Tietze [52]. Immediately after blood collection into the EDTAcontaining plastic tubes, 100 µL of each whole blood sample for GSSG determination was transferred to a 1.5-mL microtube (Eppendorf, Hamburg, Germany) containing 10 µL of the scavenger M2VP (1-methyl-2-vinyl-pyridium-trifluoromethane sulfonate) to prevent oxidation of GSH to GSSG during sample preparation. Next, 50 µL of the whole blood sample for GSH determination was transferred into a 1.5-mL microtube (Eppendorf) without treatment. All samples were frozen at −80 • C until analysis. Before analysis, blood samples were thawed and mixed. After all procedures and reaction with Ellman's reagent (5,5 -dithiobis-2-nitrobenzoic acid, DTNB), samples were measured in duplicate by using a spectrophotometric reader at 412 nm (Epoch, BioTek, Santa Clara, CA, USA). The antioxidant status of the animals was assessed by blood GSH and GSSG concentrations and GSH/GSSG ratio. The concentrations of GSH and GSSG are expressed in µmol/L. The GSH/GSSG ratio was calculated according to the instructions of the assay kit.

Statistical Analysis
SPSS software was used for statistical analysis. Data are presented as mean ± standard error of the mean (SEM). Multiple measurements from each individual were averaged and used as a single value for analysis. The Shapiro-Wilk test was applied to test the normality of the data distribution. Oxidative stress data (GSH, GSSG) were tested to identify and eliminate outliers (Grubbs' test). A Student t-test (for normally distributed data sets) or Mann-Whitney tests (for nonnormally distributed datasets) were used to determine differences between exposed and control groups. Differences with p < 0.05 were considered statistically significant. P values are given as follows: * p < 0.05; ** p < 0.01; *** p < 0.001.

Phagocytic Activity of Blood Monocytes, Granulocytes, and Respiratory Burst
Phagocytic activity of cells was evaluated by flow cytometry. TiO 2 NP exposure significantly stimulated the phagocytic activity of monocytes in mice exposed to the low dose compared with controls ( Figure 3). No significant differences in phagocytic activity of granulocytes and respiratory burst of granulocytes were recorded.
SPSS software was used for statistical analysis. Data are presented as mean ± standard error of the mean (SEM). Multiple measurements from each individual were averaged and used as a single value for analysis. The Shapiro-Wilk test was applied to test the normality of the data distribution. Oxidative stress data (GSH, GSSG) were tested to identify and eliminate outliers (Grubbs' test). A Student t-test (for normally distributed data sets) or Mann-Whitney tests (for nonnormally distributed datasets) were used to determine differences between exposed and control groups. Differences with p < 0.05 were considered statistically significant. P values are given as follows: * p < 0.05; ** p < 0.01; *** p < 0.001.

Phagocytic Activity of Blood Monocytes, Granulocytes, and Respiratory Burst
Phagocytic activity of cells was evaluated by flow cytometry. TiO2 NP exposure significantly stimulated the phagocytic activity of monocytes in mice exposed to the low dose compared with controls ( Figure 3). No significant differences in phagocytic activity of granulocytes and respiratory burst of granulocytes were recorded. Figure 3. Phagocytic activity and respiratory burst of leukocytes. Phagocytic activity of monocytes and granulocytes in the blood of mice exposed to TiO2 NPs for seven weeks was evaluated by using ingestion of fluorescein-labeled Staphylococcus aureus, and the respiratory burst was monitored by using hydroethidine by flow cytometry. Control, control group (n = 10); LD-TiO2 NPs, group exposed to the low dose of TiO2 NPs (0.012 µg TiO2/g b.w.) (n = 7); HD-TiO2 NPs, group exposed to the high dose of TiO2 NPs (0.958 µg TiO2/g b.w.) (n = 10). Results are expressed as the percentage of phagocytic activity and respiratory burst. Bars indicate mean group activity of blood cells (mean + SEM). Significance: ** p < 0.01.

Phenotypic Analysis of Spleen, Thymus, and Bone Marrow
Phenotypic analysis of cells in the spleen, thymus, and bone marrow was performed by flow cytometry. The results of the measurement of CD3 + (T-lymphocytes), CD3 + CD4 + (T-helper lymphocytes), CD3 + CD8 + (T-cytotoxic lymphocytes), CD3 − CD19 + (B-lymphocytes), and CD3 − CD335 + (natural killer (NK) cells) are summarized in Table 1. The effect of seven-week inhalation of TiO2 NPs was manifested by a significantly decreased percentage of T-lymphocytes in the spleen in both dose groups vs. controls. In mice exposed . Phagocytic activity and respiratory burst of leukocytes. Phagocytic activity of monocytes and granulocytes in the blood of mice exposed to TiO 2 NPs for seven weeks was evaluated by using ingestion of fluorescein-labeled Staphylococcus aureus, and the respiratory burst was monitored by using hydroethidine by flow cytometry. Control, control group (n = 10); LD-TiO 2 NPs, group exposed to the low dose of TiO 2 NPs (0.012 µg TiO 2 /g b.w. ) (n = 7); HD-TiO 2 NPs, group exposed to the high dose of TiO 2 NPs (0.958 µg TiO 2 /g b.w. ) (n = 10). Results are expressed as the percentage of phagocytic activity and respiratory burst. Bars indicate mean group activity of blood cells (mean + SEM). Significance: ** p < 0.01.

Phenotypic Analysis of Spleen, Thymus, and Bone Marrow
Phenotypic analysis of cells in the spleen, thymus, and bone marrow was performed by flow cytometry. The results of the measurement of CD3 + (T-lymphocytes), CD3 + CD4 + (Thelper lymphocytes), CD3 + CD8 + (T-cytotoxic lymphocytes), CD3 − CD19 + (B-lymphocytes), and CD3 − CD335 + (natural killer (NK) cells) are summarized in Table 1. The effect of seven-week inhalation of TiO 2 NPs was manifested by a significantly decreased percentage of T-lymphocytes in the spleen in both dose groups vs. controls. In mice exposed to the low dose of TiO 2 NPs, the percentage of T-helper lymphocytes in the spleen was also reduced compared with controls. The percentages of splenic CD3 + CD8 + , CD3 − CD19 + , and CD3 − CD335 + cells in exposed mice were not different from those of controls. Phenotypic analysis of the thymus and bone marrow showed no significant difference between the exposed and control mice or between the low-dose and high-dose groups for any of the parameters examined. Organs were derived from mice exposed to TiO 2 NPs for seven weeks and controls. Cells were labeled with fluorescent monoclonal antibodies and analyzed by using flow cytometry. Control, control group (n = 10); LD-TiO 2 NPs, group exposed to the low dose of TiO 2 NPs (0.012 µg TiO 2 /g b.w. ) (n = 7-10); HD-TiO 2 NPs, group exposed to the high dose of TiO 2 NPs (0.958 µg TiO 2 /g b.w. ) (n = 10). CD3 + , T-lymphocytes; CD3 + CD4 + , Thelper lymphocytes; CD3 + CD8 + , T-cytotoxic lymphocytes; CD3 − CD19 + , B-lymphocytes, CD3 − CD335 + , NK-cells.
Results are expressed as the mean group percentage of labeled cells (mean ± SEM). Significance: * p < 0.05.

In Vitro Production of Cytokines
The in vitro production of several key cytokines was examined by the luminescence method. Cytokine levels measured in cultures from spleen cells of mice are shown in Table 2. A seven-week inhalation of TiO 2 NPs in mice resulted in a significant decrease in the levels of IL-4 and IL-18 in the high-dose group compared with controls. Moderately reduced levels of IL-2, IL-10, IL-17A, IFN-γ, IL-6, TNF-α, GM-CSF, IL-13, and the chemokines MIP-1α, MIP-1β, MIP-2, and RANTES were found in mice receiving a high dose. In mice receiving a low dose of NPs, a marked but nonsignificant increase in the secretion of monocyte chemoattractant proteins (MCPs) MCP-1 and MCP-3 was found. No differences in the levels of IL-12p70, eotaxin, and IP-10 were observed. Cytokines were measured by using the luminescence method. Control, control group (n = 5-9); LD-TiO 2 NPs, group exposed to the low dose of TiO 2 NPs (0.012 µg TiO 2 /g b.w. ) (n = 4-7); HD-TiO 2 NPs, group exposed to the high dose of TiO 2 NPs (0.958 µg TiO 2 /g b.w. ) (n = 5-10). Results are expressed in pg/mL as mean group levels of cytokines (mean ± SEM). IL, interleukin; IFN, interferon; TNF, tumor necrosis factor; GMS-CSF, granulocytemacrophage colony-stimulating factor; eotaxin, eosinophil chemotactic protein; MIP, macrophage inflammatory protein; RANTES, regulated on activation normal T-cell expressed and secreted; IP, interferon gamma-induced protein; MCP, monocyte chemotactic protein. Significance: * p < 0.05.

Antioxidant Status of Blood-Reduced Glutathione and Oxidized Glutathione
The glutathione system is one of the defense mechanisms against the effects of xenobiotics and free radicals. The overall status of antioxidant protection of the organism was evaluated by GSH and GSSG content, and GSH/GSSG ratio in blood samples. The experimental group exposed to the low concentration of TiO 2 NPs showed a 32% increase in GSH concentration compared with the control group (1 037.6 ± 469.2 vs. 785.5 ± 312.3 µmol/L), but the difference was not significant. In the high-dose animals, a 74% increase in GSH concentration (1 368.6 ± 306.8 µM) was statistically significant (p = 0.006) (see Table 3). On the other hand, no significant effect on GSSG content was observed in the low-dose and high-dose experimental groups compared with the control group. Regarding the GSH/GSSG ratio, a significant dose-dependent increase was observed. 1368.6 ± 108.5 ** GSSG 60.3 ± 3.5 61.5 ± 7.0 61.4 ± 5.1 GSH/GSSG 5.3 ± 0.7 7.6 ± 1.1 * 10.5 ± 1.1 ** Blood was derived from mice exposed to two different doses of TiO 2 NPs and controls. Control, control group (n = 8); LD-TiO 2 NPs, group exposed to the low dose of TiO 2 NPs (0.012 µg TiO 2 /g b.w. ) (n = 8); HD-TiO 2 NPs, group exposed to the high dose of TiO 2 NPs (0.958 µg TiO 2 /g b.w. ) (n = 8). GSH, reduced glutathione; GSSG, oxidized glutathione. Results are expressed in µmol/L as mean values of GSH and GSSG (mean ± SEM). Significance: * p < 0.05, ** p < 0.01.

Discussion
Our study extends the number of inhalation studies on TiO 2 NPs by using a wholebody inhalation chamber model, which is otherwise extremely rare (see Supplementary  Table S1) [7,53]. In addition, none of the previous studies addressed the effects on the immune response in exposed animals. In this study, the profile of lymphocyte markers, functional immunoassays, and markers of antioxidant defense were selected to evaluate the potential adverse effects of inhalation of TiO 2 NPs in mice exposed for seven weeks. Many of the immunotoxic effects of engineered nanomaterials are mediated by direct interaction with the innate immune system [54]. Macrophages play a critical role in immune surveillance of pathogens and clearance of inhaled particles and fibers [55]. In our study, a seven-week inhalation of TiO 2 NPs stimulated the phagocytic activity of monocytes and substantially, but not significantly, enhanced the secretion of monocyte chemoattractant proteins MCP-1 (200%) and MCP-3 (167%) in low-dose mice. Activation of phagocytic activity of monocytes suggests significant action to clean NPs from the organism. Active phagocytosis of the TiO 2 NPs was confirmed by cytoplasmic proteome analysis in macrophages derived from human monocytes [56].
In addition to interfering with phagocytosis, damage to the pulmonary macrophages may be manifested by decreased chemotactic ability and MHC-class II expression on the cell surface, as well as increased secretion of nitric oxide and upregulation of inflammatory proteins (MIP and MCPs) after intratracheal instillation of TiO 2 NPs [18,35,57].
Oxidative stress and cytotoxicity seem to be the underlying mechanisms of the effect of TiO 2 NPs on macrophages. The observed oxidative stress was associated with the formation of intracellular ROS [56,58]. Cytotoxicity resulted in changes in the molecular patterns of proteins [56,58] and nucleic acids [58], remodeling of the cytoskeleton [56,59], damage to organelles [58], and formation of large vacuoles [59].
The assessment of the acquired immune response in our study included subpopulations of lymphocytes and NK cells in spleen, thymus, and bone marrow. The analysis showed a significant reduction in the percentage of T-cells in the splenic lymphocytes in both TiO 2 NPs exposed groups. T-helper cells were significantly decreased only in the low-dose mice. The depletion of T-cells suggests that exposure to TiO 2 NPs may lead to immunosuppression, as the immune system fails to respond with an increase in specific T-cell populations [60,61].
The complex functions of immune cells present in the spleen were studied by in vitro production of cytokines and chemokines. The suppressed percentage of T-lymphocytes and T-helper cells directed our attention to the production of IL-2, IL-10, and IL-17A. Indeed, the levels of all three cytokines were moderately decreased in the high-dose group of mice (63%, 60%, 48%). The decrease in IL-17A was close to statistical significance (p = 0.063). Moreover, the reduced IL-4 together with the moderately decreased lymphocytedriven cytokines IFN-γ (40%), GM-CSF (39%), IL-13 (44%), and chemokine RANTES (63%, p = 0.056) observed in mice exposed to the high dose of TiO 2 NPs is a clear indication of the toxicity of TiO 2 NPs. The overall picture is complemented by the suppressed secretory function of macrophages, as evidenced by significantly lower levels of the proinflammatory cytokine IL-18, accompanied by moderately decreased levels of TNF-α (57%), IL-6 (46%), MIP-1α (60%), MIP-1β (52%), and MIP-2 (70%) in the splenic supernatants of the high-dose animals. Similar to our results, published data on cytokine production induced by TiO 2 NPs in a long-term inhalation study in rats showed a decrease in blood IFN-γ and TNF-α levels [62]; however, no changes [8] or stimulation [34,35,63] of cytokine expression/release were observed.
It is evident that TiO 2 NPs can modulate the immune response through multiple pathways with different results [30,[64][65][66]. The suppressive effects of TiO 2 nanostructure materials may be mediated by impairment of lymphocyte development [30] and proliferation [30,66] with suppression of Th1-cytokines [66] or decrease in inflammatory-related gene transcription [63]. Finally, the suppressive effects of TiO 2 nanomaterials in the context of the immune response are best demonstrated by using animal models of complex host resistance to infection and tumors. Exposure to TiO 2 NPs exacerbated pneumonia in mice infected with respiratory syncytial virus [67] and significantly increased tumor growth in mice implanted with B16F10 melanoma cells [30].
We also examined the overall antioxidant status of the organisms and showed a dosedependent increase in GSH concentration in the whole blood of mice exposed to TiO 2 NPs. The significantly increased GSH levels may indicate the self-regulation of some enzymes and antioxidants as a defense response to oxidative stress stimulated by exposure to TiO 2 NP. Glutathione is one of the most important antioxidants abundantly present in cells and biological fluids throughout the body. The reduced form of glutathione reacts with ROS to form GSSG, which is rapidly regenerated.
Similar to our results, Wang et al. [68] reported an increase in GSH levels in the rat synovium after intraarticular injection of TiO 2 NPs. They also observed upregulated GSHperoxidase, superoxide dismutase, and lipid peroxidation. In a short-term study, Sangeetha et al. [69] found increased GSH content and lipid peroxidation in the liver and spleen of mice treated orally with 1.6 mg/kg b.w. of TiO 2 NPs. Some in vitro studies have also shown elevated GSH levels after TiO 2 NP exposure [70,71]. The effect of TiO 2 NP exposure in human bronchial epithelial cells (increase or decrease in GSH level) depended on the dose, size, and agglomeration of TiO 2 NPs [71].
On the other hand, our results are not consistent with the results of other subchronic inhalation studies, in which a decrease or no change in GSH levels in blood or tissues was observed. However, the doses administered in these experiments were much higher compared to our study. Liu et al. [62] found decreased GSH levels in the blood of rats after intratracheal instillation of TiO 2 NPs (3.5 and 17.5 mg/kg b.w. ), whereas Relier et al. [72] reported no change in total glutathione levels in the plasma, lung, and liver of rats after intratracheal exposure to TiO 2 NPs (0.5, 2.5 and 10 mg/kg b.w. ). Wang et al. [73,74] also observed no change in GSH levels in the brain of female mice intranasally administered 500 µg of a TiO 2 NP suspension every other day for 20 or 30 days, whereas GSH levels were significantly increased 10 days after exposure.
We suppose that the increase in GSH content observed in the present study may be a response to the excessive formation of ROS, as defense mechanisms were activated due to TiO 2 NP exposure and GSSG reduction was enzymatically increased in favor of GSH. Considering the low dose of TiO 2 NPs used in our study, we suppose that the increased GSH levels were likely the primary means by which cells prevented lipid hydroperoxide formation. The enhancement of enzymatic reactions and the increase in GSH production could be a triggering mechanism to support defense against the deleterious effects of TiO 2 NPs. This finding is in agreement with the results of Carmo et al. [75] who observed increased GSH content and no change in antioxidant enzyme activity in fish liver after acute exposure (48 h) to 1, 5, 10, and 50 mg/L TiO 2 NPs, whereas subchronic exposure (14 days) to the same TiO 2 NP concentrations decreased superoxide dismutase activity and increased glutathione-S-transferase activity and GSH content. Nevertheless, the animal strain and genetic background used, the dose and duration of exposure, and the size and agglomeration of TiO 2 NPs may affect the antioxidant defense of the organism.
We exposed animals to TiO 2 NPs in whole-body inhalation chambers to simulate natural conditions. In this regard, it should be mentioned that in this procedure TiO 2 NPs may adhere to the walls of the polycarbonate boxes in the inhalation cages or come into contact with the food or various body parts of the mice such as the respiratory tract, the olfactory system, the fur or skin of the mouse, and also the eyes. As a result, mixed inhalation and oral uptake of nanoparticles is possible. To minimize the oral intake of TiO 2 NPs, the feed was placed in a special feeding device that we designed to protect the granules from nanoparticle contamination. However, mutual licking of the fur by the animals cannot be avoided. Licking of nanoparticles adhering to the walls of the boxes was minimized by cleaning the walls during regular feed replenishment and bedding changes.

Conclusions
TiO 2 NPs are used in large quantities in various industries worldwide, and therefore experimental studies on possible toxic effects are important to ensure human safety. Our results showed a dose-dependent effect of TiO 2 NPs by inhalation on innate immunity, which was manifested by stimulated phagocytic activity of monocytes in low-dose mice and suppressed secretory function of monocytes in high-dose animals. The effect of TiO 2 NPs on adaptive immunity, which was manifested in the spleen by a decrease in the percentage of T-cells, a reduction in T-helper cells, and a dose-dependent decrease in cytokine production by lymphocytes, may indicate immunosuppression. The dosedependent increase in GSH concentration and GSH/GSSG ratio suggests self-regulation of some enzymes and antioxidants as a defence response to oxidative stress stimulated by TiO 2 NP exposure.
In summary, our results show that relatively low doses of TiO 2 NPs have significant immunomodulatory effects in mice affecting innate and adaptive immune responses. This indicates the potential risk of adverse health effects from inhalation of TiO 2 NPs. This information may be useful for risk assessment of exposure to TiO 2 NPs.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/nano13040767/s1, Table S1: In vivo inhalation studies investigated the adverse effects of TiO 2 NPs on systemic immune response and oxidative stress. References [76][77][78] are cited in the supplementary materials.

Data Availability Statement:
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

Acknowledgments:
We thank Helena Nagyova and Edita Mrvikova for technical assistance and Andrew Collins for critical reading and language corrections of the manuscript. The authors would like to thank F. Mika (Institute of Scientific Instruments of the Czech Academy of Sciences) for help with the processing of samples.

Conflicts of Interest:
The authors declare no conflict of interest.